Source of the catalytic differences between human carbonic anhydrases III and II as determined by site-specific mutagenesis

152 Pages·1992·4.4 MB·English

by  LoGrasso, Philip Victor

SOURCE OF THE CATALYTIC DIFFERENCES BETWEEN HUMAN CARBONIC
ANHYDRASES III AND II AS DETERMINED BY SITE-SPECIFIC
MUTAGENESIS
BY
PHILIP VICTOR LOGRASSO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1992
—
To my father for teaching me his principles caring,
:
dedication, and honesty
—
To my mother for her unconditional love
—
To my friends for the good times
. .
ACKNOWLEDGEMENTS
I am very grateful to many people for helping me in my
work. Dr. David Silverman has been a true mentor. He has
taught me to be a thorough and diligent scientist The data
.
analysis, writing, and organizational skills have learned
I
from him are invaluable
Dr. C.K. Tu warrants a special mention because a great
many problems were solved with a simple, "Hey Tu " I have
.
learned a great deal about enzyme kinetics from Tu, but it is
his generous spirit and willingness to help that I most hope
I retain. Thanks Tu
Three people have been a great help on the molecular
biology aspect of this work: Drs Phil Laipis, Susan
.
Tanhauser, and David Jewell. Each has contributed to my
knowledge in this field, and for that I am grateful.
George Wynns taught me a great deal about protein
purification and deserves many thanks. Our conversations
about baseball, Florida, and life always brightened the day.
It was also a pleasure working with all of the staff in the
Pharmacology Department. Special thanks go to Judy Adams for
formatting this thesis and all her help.
Finally, no acknowledgement would be complete without
thanking my parents and friends for their encouragement and
love
.
111
TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS ix
ABSTRACT x
CHAPTER INTRODUCTION
1 1
:
Investigating the Catalytic Mechanism of Carbonic
Anhydrase using Site-Directed Mutagenesis
1
Genetic Differences and Tissue Distribution of
Various Carbonic Anhydrase Isozymes 5
Biological Role of Carbonic Anhydrase III 7
Structure of Carbonic Anhydrase 8
General Shape of the Molecule 9
Secondary Structure 12
Catalytic Mechanism 18
Interconversion of CO2 and HCO3- 19
Proton Transfer 20
Dehydration 21
CHAPTER 2 THE MUTAGENESIS, EXPRESSION, PURIFICATION,
:
AND KINETIC METHODS USED TO CREATE AND CHARACTERIZE
HCA III MUTANTS 25
Mutagenesis 25
Growth of Phage 29
Isolation of ss template DNA 30
Phosphorylation and Hybridization of the
Site-Specific Oligonucleotide 31
In Vitro Double Strand DNA Synthesis 32
Screening Potential Mutants 32
Cassette Mutagenesis 33
Creation of Remaining Mutants 35
Expression of HCA III Mutants in E coli 35
Purification of Human Carbonic Anhydrase Mutants
Expressed in E coli 42
Steady-State Kinetic Methods 52
Stopped-Flow Spectrophotometry 52
4-Nitrophenyl Acetate Hydrolysis 54
Equilibrium Kinetic Methods 58
IV
CHAPTER 3 CATALYTIC ENHANCEMENT OF HUMAN CARBONIC
:
ANHYDRASE III BY REPLACEMENT OF PHE 198 WITH LEU 61
Introduction 61
Results 62
Discussion 77
Interconversion of CO2 and HCO3- 77
Proton Transfer 81
Inhibition 84
Conclusion 86
CHAPTER 4 EFFECTS OF THE MOLECULAR PROPERTIES OF
:
RESIDUE 198 ON CATALYSIS AND INHIBITION OF HCA III ....87
Introduction 87
Results 88
Discussion 98
Interconversion of CO2 and HC03~ 100
Proton Transfer Ill
Inhibition 121
Conclusion 123
CHAPTER 5 DISCUSSION AND CONCLUSIONS 125
:
LIST OF REFERENCES 132
BIOGRAPHICAL SKETCH 138
v
,
LIST OF TABLES
Table Page
1. Comparison of maximal steady-state rate constants
and values of the apparent pK for the hydration
a
of CO2 and the hydrolysis of 4-nitrophenyl
acetate catalyzed by HCA III and HCA II 3
2. Comparison of amino acid differences near the
active sites of HCA III and HCA II 11
3. Maximal (pH-independent steady-state constants
)
and values of apparent pK for the hydration of
a
CO2 and the hydrolysis of 4-nitrophenyl acetate
catalyzed by carbonic anhydrase and mutants 63
4. Inhibition constants Kj (micromolar) determined
from Ri, the catalyzed rate of interconversion
of CO2 and HCO3- at chemical equilibrium 75
5. Solvent hydrogen isotope effects on Ri and RH20
catalyzed by HCA III, HCA II, and two mutants 76
6. Maximal (pH-independent steady-state constants
)
and values of the apparent pK for the hydration
a
of CO2 and rate of release of H218 catalyzed by
mutant and wild-type carbonic anhydrases 89
7. Inhibition constants Kj (micromolar, ethoxzolamide)
(millimolar, cyanate) determined from Ri, the
,
catalyzed rate of interconversion of CO2 and HCC>3~
at chemical equilibrium 99
VI
LIST OF FIGURES
Figure page
1 Comparison of amino acid sequences for human
CA II and CA III 10
2 X-ray crystal structure of part of the active
site cavity of bovine carbonic anhydrase 13
3 Side-by-side representation of some of the amino
acid side chains found in the active site cavity of
carbonic anhydrase III and II 16
4 Oligonucleotide-directed mutagenesis using single-
stranded template containing uracil residues 28
5 Location of key features on the pET81fl HCA III
expression vector 38
6 Time course of F198V HCA III synthesis after
induction with 40 |IM IPTG 41
7 Flow chart detailing the purification of human
carbonic anhydrase III mutants expressed in E coli 44
8 Elution profile of K64H-R67N HCA III after
purification on DEAE-sephacel 48
9 UV absorbance spectrum for purified F198A HCA III 49
10 12% polyacrylamide gel detailing the levels of purity
of K64H-R67N HCA III during the different stages of
carbonic anhydrase purification 51
11 Typical data analysis used to calculate steady-state
kinetic constants 56
12 Carbonic anhydrase catalyzed 180-exchange at chemical
equilibrium
60
13 Comparisons of kcat/Km for hydration of CO2 catalyzed
by variants of HCA III obtained by site-directed
mutagenesis at positions 64, 67, and 198 in the
active site cavity of HCA III 66
Vll
14 The pH dependence of RH20/ [E] for F198L HCA III;
R67N-F198L HCA III; and K64H-R67N-F198L HCA III 69
15 Comparison of the water-off rates, RH20/tE for
]
variants of HCA III at positions 64, 67, and 198
in the active site cavity of HCA III 71
16 The dependence on the imidazole concentration of
RH20/[E] and Rl/[EJ catalyzed by F198L HCA III;
R67N-F198L HCA III; and K64H-R67N-F198L HCA III 73
17 The pH dependence of the logarithm of kcat/Km for
the hydration of CO2 catalyzed by F198D HCA III,
and F198N HCA III 92
18 The pH dependence of the logarithm of kcat for the
hydration of CO2 catalyzed by F198D HCA III, and
F198N HCA III 94
19 The pH dependence of RH20/ [E] for F198D HCA III,
and F198N HCA III 97
20 Plot of the logarithm of kcat/Km for CO2 hydration
versus pKa of zinc-bound water for six amino acid
replacements at position 198 and for wild-type
HCA III 102
21 Plot of the logarithm of kcat/Km for CO2 hydration
versus the logarithm of Ki0CN~ for six amino acid
replacements at position 198 and for wild-type
HCA III 107
22 Plot of the logarithm of kcat versus AG transfer
of the amino acid side chain from nonaqueous
solution to water 118
Vlll
-
LIST OF ABBREVIATIONS
BCA bovine carbonic anhydrase
BSA bovine serum albumin
CA carbonic anhydrase
CD circular dichroism
CDNA complimentary DNA
CHES 2 [N-cyclohexyl amino] ethanesulfonic acid
DEAE-sephacel diethylaminoethyl sephacel
DNA deoxyribonucleic acid
DNase deoxyribonuclease
dsDNA double-stranded DNA
DTT dithiothreitol
EDTA ethylenediaminetertaacetic acid
g gravitational force
HCA human carbonic anhydrase
HEPES N-2-hydroxyethylpiperazine-N
'
2-ethanesulfonic acid
IPTG isopropylthio-Ji-D-galatopyranoside
kDa kilodalton
LB Luria broth
MOPS 3- (N-morpholino)propanesulfonic acid
MW molecular weight
NMR nuclear magnetic resonance
OD optical density
PEB phenol extraction buffer
PEG polyethylene glycol
SDS sodium dodecyl sulfate
SSC standard sodium citrate buffer
ssDNA single-stranded DNA
TAPS N-{ [Tris (hydroxymethyl) methyl] amino}
propane sulfonic acid
TBE tris-borate EDTA
Tris tris (hydroxymethyl) aminomethane
ix
.
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SOURCE OF THE CATALYTIC DIFFERENCES BETWEEN HUMAN CARBONIC
ANHYDRASES III AND II AS DETERMINED BY SITE-SPECIFIC
MUTAGENESIS
By
Philip V. LoGrasso
May, 1992
Chairman: David N. Silverman
Major Department: Pharmacology and Therapeutics
The objective of this work was to determine which of the
unique amino acids near the active site of the low activity
human carbonic anhydrase (HCA III) is the molecular source of
the 500-fold difference in catalytic and inhibition
properties between HCA III and human carbonic anhydrase II
(HCA II)
Mutants of HCA III were made by oligonucleotide-directed
mutagenesis replacing three residues near the active site
with amino acids known to be at the corresponding positions
in HCA II (Lys 64 -» His, Arg 67 -> Asn, and Phe 198 ->Leu).
Emphasis was placed on Phe-198 where replacement residues
varied in size, charge, and hydrophobicity and were selected